Overview. Plant biotechnologists have, because of the toxic byproducts found in castor bean (Ricinus communis), labored for years to produce a temperate oilseed crop that produces triacylglycerols rich in ricinoleic acid, which is the active component of castor oil. However, these labors, including the singular expression of the castor hydroxylase enzyme in Arabidopsis, or expression of FAH12 in transgenic tobacco or Arabidopsis, has not been sufficient to provide for high level accumulation of ricinoleate. There is, therefore, a pronounced need in the art for novel compositions and methods to enhance hydroxy fatty acid in plants (e.g., oilseed plants), and particularly in temperate oilseed crops (e.g., soybean or canola).
Limited Source of Ricinoleic acid. Ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: 18:1-OH) is a naturally-occurring compound with great value as a petrochemical replacement in a variety of industrial processes. Its derivatives are found in products such as dyes, lubricants, nylon, soaps, inks, adhesives, and bio-diesel1. Ricinoleic acid is produced in a very limited number of plant species, with the primary source being the seeds of castor bean (Ricinus communis). Ricinoleate makes up approximately 90% of the total castor seed fatty acids, primarily in the form of triacylglycerol (TAG). Castor oil is produced commercially from undomesticated plants grown in tropical climates2. Castor bean cannot be agriculturally optimized to accommodate demand, and it is therefore desirable to use a temperate oilseed crop, such as soybean or canola, as a platform for transgenic production of ricinoleate-rich oils.
Involvement of Oleoyl-12-Hydroxylase in the Natural Biosynthesis of Ricinoleic acid. Ricinoleic acid is formed by a hydroxylase enzyme that adds a hydroxy group to the twelfth carbon of oleic acid moieties esterified to the sn-2 position of phosphatidylcholine (PC)3,4. This reaction requires a cytochrome b5 electron donor and molecular oxygen5,6 and takes place in the endoplasmic reticulum (ER) membrane5, which presumably allows for efficient channeling of ricinoleic acid into triacylglycerol (TAG) within the ER.
Prior Art Attempts to Increase Ricinoleic Acid Accumulation. There have been a number of prior art attempts to enhance accumulation of ricinoleic acid, including: (A) identification and expression of castor hydroxylase enzyme in FAH12 in transgenic tobacco or Arabidopsis; and (B) use of mutants deficient in FAD2 activity.
(A) Identification and Over-expression of Castor Hydroxylase Enzyme FAH12. Additionally, and based on shared biochemical characteristics (e.g., use of the same 18:1 substrate) between the castor hydroxylase and the broader family of fatty acyl desaturases3,6, van de Loo et al.7 screened a castor bean developing endosperm cDNA library for sequences homologous to the desaturases. A cDNA clone (named FAH12) was identified whose predicted protein shared approximately 67% amino acid identity with Arabidopsis FAD2, the enzyme that catalyzes the desaturation of oleate (18:1) to linoleate (18:2). Unfortunately, expression of FAH12 in transgenic tobacco caused the accumulation of ricinoleic acid, but only to very low levels7.
Several laboratories have since attempted to identify and overcome the limitations to high-level production and accumulation of ricinoleic acid in plants. For example, Arabidopsis has shown great promise as a model system plant for studying castor seed oil biosynthesis, and seed-specific overexpression of FAH12 in Arabidopsis has resulted in higher ricinoleate levels than seen in tobacco. Unfortunately, however, the highest amount of hydroxy fatty acid accumulation in these Arabidopsis lines represented approximately 17% of total seed lipid8,9, far below the ˜90% found in castor bean and certainly less than would be necessary for practical use as a castor oil replacement.
When Arabidopsis was transformed with the hydroxylase cDNA, four novel hydroxy fatty acids were found to accumulate in the seeds (Broun and Somerville, 1997). In addition to ricinoleic acid accumulation, densipolic acid (12-hydroxy-octadec-cis-9,15 enoic acid: 18:2-OH), lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid: 20:1-OH), and auricolic acid (14-hydroxy-eicos-cis-11,17-enoic acid: 20:1-OH) accumulated to a small degree. These latter three fatty acids are not found to accumulate in castor bean seeds, but do accumulate in another hydroxy fatty acid producing species, the Lesquerella species. Members of the Lesquerella species each distribute their hydroxy fatty acids differently, but almost all species contain their hydroxy fatty acids as densipolic, lesquerolic, or auricolic acids instead of ricinoleic acid (Hayes et al., 1995). These three hydroxy fatty acids are also chemically valuable and the subspecies Lesquerella fendleri is grown in some areas as a seed oil crop (Abbott et al., 1997). It is thought that transgenic Arabidopsis lines metabolize ricinoleic acid similarly to the Lesquerella species since they both produce all four of these hydroxy fatty acids (Broun et al., 1998). A putative pathway suggests that the Arabidopsis fatty acyl desaturase, FAD3, is responsible for the desaturation of 18:1-OH to 18:2-OH, while fatty acyl elongase 1, FAEI, elongates both 18:1-OH and 18:2-OH to 20:1-OH and 20:2-OH, respectively (Broun and Somerville, 1997). Although the presence of these additional three hydroxy fatty acids contribute to the total seed hydroxy fatty acid in transgenic Arabidopsis lines, the total amount of accumulation has not breached the 20% hydroxy fatty acid mark.
(B) Use of Mutants Deficient in FAD2 Activity. When hydroxy fatty acids accumulate in Arabidopsis seeds, the amount of non-hydroxy 18:1 and 18:2 deviate considerably from wild-type amounts as summarized in Table 1:
TABLE 1Typical fatty acid profile seen for wild-type Columbia seed (Col) versus seedtransformed with the castor bean hydroxylase cDNA (CBH).Plant Name16:018:018:118:218:320:118:1-OH18:2-OHCBH9.6%4.1%25.3%19.9%9.7%16.2%8.8%6.4%Col8.5%3.1%17.1%31.3%21.6%18.1%0.0%0.0%
The accumulation of 18:1 increases while 18:2 decreases. This divergence from wild-type levels has been speculated to be the result of inhibitory effects on the FAD2 desaturase by the hydroxylase, either directly or indirectly (Broun and Somerville, 1997). The inhibitory effects could hinder 18:1 from being used by either FAD2 or the hydroxylase, thus causing levels of 18:1 to increase past wild-type levels. Prior art attempts were therefore made to transform the hydroxylase cDNA into an Arabidopsis mutant deficient in FAD2 activity, thereby eliminating these inhibitory effects. However, when this experiment was performed, the levels of 18:1-OH did not increase significantly (Smith et al., 2003), indicating that the amount of 18:1 substrate is not a limiting factor for hydroxylase activity in the transformed Arabidopsis lines.
There is therefore, a pronounced need in the art for modifying plant oils, including provision of alternative crop sources for certain oils products and/or means to provide novel fatty acid compositions and/or accumulations for plant seed.